Long-term environmental enrichment prevents schizophrenia-like abnormalities and promotes hippocampal Slc6a4 gene demethylation in mice submitted to a two-hit model

doi:10.21203/rs.3.rs-2973577/v1

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Long-term environmental enrichment prevents schizophrenia-like abnormalities and promotes hippocampal Slc6a4 gene demethylation in mice submitted to a two-hit model

https://doi.org/10.21203/rs.3.rs-2973577/v1

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In the last decades, attention has been called to the need for preventive strategies against neurodevelopmental disorders. In the present study, we aimed to investigate the impacts of long-term environmental enrichment (EE) in preventing behavioral, neurochemical, and epigenetic changes in mice exposed to the two-hit model of schizophrenia. To this end, we used neonatal Swiss mice exposed to the viral mimetic polyinosinic:polycytidylic acid (PolyI:C) as first-hit on postnatal days (PND) 5–7 or sterile saline (zero-hit). On PND21, mice were randomly allocated to cages with standard (SE) or enriched environment (EE). From PND35-44, PolyI:C group was exposed to unpredictable stressors as second-hit. On PND70, after EE's last exposure, the animals underwent behavioral testing, and the hippocampus was collected for biochemical (Iba-1 and DCX) and epigenetic (SLC6A4 gene) analysis. The results showed that fifty days of EE exposure to two-hit mice, i.e., from infancy to adulthood, prevented sensorimotor gating deficits and working memory impairment while improving locomotor and exploratory activity. Furthermore, EE prevented hippocampal Iba-1 increased expression. EE-exposed mice presented increased hippocampal DCX expression. In addition, hippocampal demethylation of the SLC6A4 gene (serotonin transporter), an epigenetic reprogramming mechanism, was observed in the two-hit group submitted to EE. Our results reveal the preventive effects of long-term EE in mice exposed to the two-hit model of schizophrenia by mechanisms related to increased neurogenesis, reduced microglia reactivity, and epigenetic regulation of serotonergic signaling.

Biological sciences/Developmental biology/Disease model

Biological sciences/Developmental biology/Neurogenesis

Biological sciences/Neuroscience/Diseases of the nervous system

Biological sciences/Neuroscience/Neuroimmunology

Schizophrenia is a mental disorder, multifactorial in origin, with highly diverse clinical manifestations ¹. This mental disorder affects approximately 0.75% of the world's population ². Based on its multifactorial origin, schizophrenia risk factors are genetic (60–80%) ³ and environmental ⁴, compromising brain development.

Concerning environmental insults, perinatal infections combined with peripubertal psychological trauma increase schizophrenia risk in adult life, especially in male subjects ⁵. This has been observed in humans ⁵ and animal studies ^6,7. For this reason, in the present study, we chose the two-hit model of schizophrenia based on neonatal exposure to a viral mimetic polyinosinic:polycytidylic acid (PolyI:C) combined with peripubertal unpredictable stress - PUS ^6,8. Indeed, perinatal exposure to PolyI:C impairs hippocampal-dependent functions, such as cognition ⁹, since this brain area is susceptible to viral-like immune activation ^9,10. Additionally, the anterior hippocampus is the key regulator of the mesolimbic dopamine system ¹¹, a key neurotransmitter in schizophrenia. It deserves mentioning that schizophrenia-like alterations induced by PolyI:C immune challenge are reversed by antipsychotic drugs ¹².

Despite dopaminergic mechanisms being the most studied in schizophrenia ¹³, serotonergic neurotransmission and, more recently, the serotonin (5-HT) reuptake pathway has gained increased evidence ¹⁴. In this regard, recent evidence points to the 5-HT transporter (5-HTT) gene (SLC6A4) functional polymorphisms as risk factors for schizophrenia ¹⁴.

In the last decades, attention has been called to the need for preventive strategies against neurodevelopmental disorders ¹⁵. To this end, understanding the epigenetic mechanisms is imperative. Moreover, as preventive strategies, non-pharmacological ones, such as environmental modulation, are welcome due to the lack of adverse effects. In this regard, adolescent environmental enrichment (for seven days) prevented schizophrenia-like alterations in the methylazoxymethanol (MAM) rat model of schizophrenia with the participation of epigenetic mechanisms ¹⁶. Also, mGlu5 KO mice, a genetic model of schizophrenia, submitted to four weeks of EE presented reduced behavioral alterations accompanied by upregulation of N-methyl-D-aspartate receptor (NMDAR) subunits ¹⁷.

Even though previous studies have found preventive effects of environmental modulation in genetic and neurodevelopmental models of schizophrenia, as far as we know, there is no evidence for a long-duration protocol (7 weeks) in the two-hit model based on perinatal immune challenge followed by peripubertal stress. Additionally, no previous study evaluated the DNA methylation profile of the 5-HTT gene (Slc6a4) as a possible mechanism underlying two-hit-induced schizophrenia and the effect of EE. Therefore, in the present study, we hypothesized that long-term EE could effectively prevent schizophrenia-like behavior in mice exposed to the two-hit model by mechanisms related to protection against hippocampal microglial proinflammatory alterations and epigenetic modulation of Slc6a4. Our primary outcome was determining the effects of EE condition against behavioral alterations induced by the two-hit model. Our secondary outcome was verifying the effects of EE condition in preventing hippocampal pro-inflammatory and Slc6a4 gene methylation.

2.1 Animals and ethical aspects

The experiments were performed in Swiss mice from the Drug Research and Development Center (NPDM) animal facility, maintained in microisolator cages (Micro-Isolator® Systems, Lab products) at 22 ± 2°C, photoperiod of 12h, and relative humidity of 60%. All animals had ad libitum access to food and water.

For the obtention of the male neonatal mice (n = 45), 12 pregnant females (who were mated a maximum of twice during the study, with different males) were monitored for the parturition day, counted as postnatal day 0 (PND0). The male pups were maintained in a microisolator cage with the dams until weaning on PND21. Female pups were used for other experiments. Sex was visually determined as described elsewhere ¹⁸. We used only two male pups from each gestation/dam randomly allocated for saline or PolyI:C administration to avoid the litter effect.

The Ethics Committee on the Use of Animals of the Federal University of Ceará approved the study (protocol 136/14). Furthermore, the experiments were performed following the resolutions from the National Council for Animal Experimentation Control (CONCEA/Brazil), the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, USA ¹⁹, and the ARRIVE guidelines reporting animal research ²⁰.

2.2 Study Design

The experiments were performed in animals allocated into four groups: zero-hit + SE (animals maintained in their home cages in a standard environment without hit exposure – considered as the control group), zero-hit + EE (animals not exposed to hits and kept in an enriched environment), two-hit + SE (animals exposed to the two-hit model maintained in a standard environment), and two-hit + EE (animals exposed to the two-hit model and maintained in an enriched environment).

For the implementation of the two-hit model, the experiments started in PND5-7, in which the animals received a single daily dose of sterile saline (zero-hit) or Polyinosinic: polycytidylic [PolyI:C, Sigma-Aldrich, St. Louis - USA (catalog number P1530)], a synthetic double-stranded RNA, as the first hit. PolyI:C was dissolved in sterile saline to yield a final 1 mg/kg/mouse dose, administered in a volume of 50 µL/8 g body weight. All solutions were freshly prepared on the day of administration. During injections, dams were removed from the home cage and returned once all pups had received the injection. The neonatal challenge caused no mortality in the puppies. The neonatal injections were performed quickly to avoid stress due to maternal separation²¹ and the litter effect ²². The use of PolyI:C in neonatal life was based on the increased risk for schizophrenia due to viral exposure in the third trimester of pregnancy ^23,24. Indeed, the correlation between mice and human neurodevelopment indicates that the degree of brain maturation observed in a pre-term and term human infant is equivalent to PND 1–10 in mice ²⁵.

The pups were maintained with their respective dams in individual cages until weaning and group separation on PND21. We performed the experiments in males since clinical studies show that men present more severe schizophrenia symptoms ²⁶. The sample size was calculated using G*Power 3.1 based on previous prepulse inhibition of the startle reflex (PPI) results obtained from mice exposed to the two-hit model and treated with candesartan as a preventive strategy ⁶. Hence, based on this previously published study ⁶, the effect size calculated based on the means of 4 experimental groups comprising 40 animals was 10.766. Considering an a error probability of 0.05, and a power of 0.95, the calculator estimated 8 animals as the total sample size for each experiment.

After weaning, male offspring from different dams of the same neonatal treatment conditions (saline or polyI:C) were randomized to an EE or SE condition. The EE consisted of a large autoclavable polypropylene cage with length [l] x width [w] x height [h] dimensions: 60 x 50 x 22 cm, and a floor area (A) equal to 3000 cm² (A = l x w). In contrast, the microisolator cage (standard housing) had 41 x 34 x 16 cm dimensions and a floor area equal to 1390 cm². Hence, dividing the floor area of the EE and SE cages by the number of animals in each cage (n = 8), in the case of EE, each animal had 375 cm2, corresponding to more than 2 times the floor area available for an animal in a standard housing cage. In addition, the EE cage presented cardboard boxes, tunnels, sheets, and wooden and plastic objects that were changed weekly ²⁷. The animals were submitted to EE from infancy, PND 21, to adulthood, PND70. Supplementary Figure S1 shows the cages used to simulate standard and enriched environmental conditions.

Subsequently, between PNDs35–43 (peripubertal phase), mice first hit with polyI:C was submitted to unpredictable stress for 5 days. The PUS protocol was based on previous studies ^6,7. The four distinct stressors are briefly described below and were applied in the following order: (i) electric foot shock; (ii) restraint stress; (iii) forced swimming stress, and (iv) water deprivation. After each stressful event, there was a one-day rest period. The first stressor event started at PND35.

PND35: Electric foot shock. Mice were exposed to three shock stimuli for 1 second each and 3 minutes for the intershock intervals. The shock intensity was set at 0.3 mA. The session ended with an additional 3-minute period, in which there was no shock stimulus.
PND37: restraint stress. Mice were horizontally immobilized for 45 minutes in the acrylic cylindrical shape, with a diameter of 27 mm × lengths of 117 mm.
PND39: forced swimming stress. The mice were placed individually in an acrylic cylinder tank (50 cm height x 18 cm diameter) containing 30 cm from the bottom of the water at room temperature. The animals were submitted to two sessions of forced swimming test, 1 minute per session, and a 3-minute break between sessions.
PND41: water deprivation. This stress was applied by removing the water bottles from the homecage at 4:00 p.m. Water consumption was restored at 8 a.m. the next day (PND42). So, the animals underwent water deprivation for 16 hours.
PND43: Another section of restraint stress.

Figure 1 presents an overview of the experimental design adopted in this study.

2.3 Behavioral tests

We conducted three behavioral determinations: the prepulse inhibition of the startle reflex (PPI) to measure sensorimotor gating, the open field test (OFT) to evaluate locomotion and exploratory capacity, the Y-maze test to assess spatial working memory, and the social interaction test (SIT). Raters blinded to the experimental groups performed the behavioral determinations. In brief, the PPI test was conducted in a startle chamber (Insight, São Paulo, Brazil) based on a previously published protocol ²⁸. The test began with a 5 min acclimatization to the startle chamber in the presence of 68 dB background noise that remained constant throughout the test session. Next, the animals received ten 120 dB single pulses with 40ms duration each and a 15-second break between pulses (individual startle amplitude). Subsequently, each mouse has then exposed to six Pulse (P) sessions (120dB, 40ms duration); six sessions of each prepulse (PP) intensity (70, 75, and 80 dB, frequency 3000 Hz, 20 ms duration), and six exposures at each prepulse intensity followed by a 120 dB startle pulse after 80 ms. The level of PPI in each mouse was determined by expressing the PP + pulse startle amplitude as a percentage decrease from pulse-alone startle amplitude, according to the following formula: %PPI = 100 × [100 × (PP/P)].

The OFT was performed in a field (30 cm × 30 cm × 15 cm) equally divided into nine parts (zones). We allocated each animal to the center of the apparatus and freely explored the field for 5 min. The parameter recorded was the traveled distance. Panlab SMART video tracking system 3.0 software (PANLAB HARVARD Apparatus) was used to record and measure the mouse exploratory activity ²⁹.

The Y-maze test was conducted in an apparatus containing three identical arms identified (arms 1, 2, and 3), interconnected at an angle of 120° to each other. Animals with preserved working memory tend to enter the unvisited arms, presenting spontaneous alternations considered correct, such as 1,2,3 − 2,3,1 or 3,2,1. Entries in repeated arms are regarded as errors. Each animal was allowed to explore the Y-maze for 8 min freely. The number of correct alternations and the percentage of correct alternations calculated were recorded ³⁰.

The SIT was performed in a three-chamber apparatus. The chambers represent a social stimulus versus a non-social stimulus. The chambers have a wire pencil cup 10.2 cm (diameter) × 10.8 cm (height). In the social chamber, there is one age-, sex-, and strain-matched unfamiliar mouse under one cup. In the non-social chamber, we included an inanimate object. The animal is placed into the apparatus containing the social- and non-social stimuli for 10 minutes. The social preference index was calculated as “time spent in the social chamber/time in social + time in the non-social chamber” ³¹.

2.4 Biochemical and methylation analysis

After behavioral evaluation, the animals were euthanized by decapitation to stop brain activity quickly. Next, the brain was removed and manually dissected on ice to collect the hippocampus. Then, the hippocampus was frozen rapidly in liquid nitrogen and stored at − 80°C until analysis.

We performed Western blot analyses to determine the expression of ionized calcium-binding adaptor molecule 1 (IBA-1) and the neuronal migration protein doublecortin, DCX. The hippocampus was homogenized in RIPA lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 5 mM EDTA, 1% NP 40, 1% triton-X-100, 1% deoxycholate 0.1% SDS) with protease inhibitor (1:100; Sigma-Aldrich, Brazil). The samples were centrifuged at 13,000 × g for 17 min at 4°C, and the supernatant was transferred to a new microtube. Total protein concentration was determined by the Bradford colorimetric method following the manufacturer's instructions (Bio-Rad Protein Assay). SDS polyacrylamide gel electrophoresis (10%) was performed using 50 µg of protein (previously prepared with a Laemmli sample buffer and heated at 95°C for 5 min). After electrophoresis, proteins were transferred to the nitrocellulose membrane, blocked with BSA 5% for 1h, and incubated overnight at 4°C with the following primary antibodies prepared in BSA 1%/TBS-T: anti-IBA (1:2000; Wako, USA), anti-DCX (1:1000; Sigma, USA), or anti-α-Tubulin (1:4000; Sigma, USA). After washing, the membranes were incubated in Horseradish peroxidase-conjugated (HSP) goat anti-mouse IgG (1:1000; Sigma, USA) for 2 h (secondary antibody). ImageQuant 300 Imager System (GE 75 Healthcare, EUA) detected the membrane protein bands. The band densities were measured with NIH Image J software n = 8 per group, chosen at random.

The genomic DNA was extracted using the ReliaPrep™ gDNA Tissue Miniprep System (Qiagen, Hilden, Germany) for high-resolution melting analysis according to the manufacturer’s instructions. The quality and quantity (260/280 and 260/230 optical density [OD] ratios) of DNA were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Rockford, IL, USA). According to the manufacturer's specifications, the bisulfite DNA modification was done with EZ DNA Methylation™ (Zymo Research). Control DNA (hypomethylated/hypermethylated) was modified by the same method as the HPC samples. Solute Carrier Family 6 Member 4 (slc6a4), the 5-HTT gene, was chosen for the HRM analysis.

The SLC6A4 gene promoter region was identified using the UCSC Genome Bioinformatics sequences, and the CpGs sites were determined by the MethyPrimer program ³². The primers were designed on the GpG island closest to the first exon, and the sequences were validated in silico using the NetPrimer program (Premier Biosoft). The primer sequences used were: SLC6A4-F 5'-TAGGAATGATGGGGGGGTTTTGT-3' and slc6a4-R 5'-CCTCACATAATCTAATCTCTAAATA-3'. The high-resolution melting (HRM) assay was performed using 1.0 µL of modified DNA from each sample, 1X of MeltDoctor HRM Master Mix (Applied Biosystems, Foster City, California), and 0.3 pmol of each primer (slc6a4 gene) in a final volume of 10 µL, in duplicate. A standard curve was performed for each experiment with the modified control DNA and different percentages of methylated DNA (0%, 50%, and 100% methylated) to mimic DNA samples with defined DNA methylation levels. Amplification reactions were performed on the LightCycler® 96 System (Roche Diagnostics) under the following conditions: initial denaturation step at 95°C for 10 minutes, 45 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 1 min, extension at 72°C for 15 s. After HRM analysis, a post-PCR method for screening unknown genetic variations was performed at the temperature and fluorescence acquisition setting recommended by the manufacturer. Normalized Melting curves were qualitatively analyzed.

2.5 Statistical analysis

All results in the figures are expressed as mean ± Standard Error of the Mean (S.E.M.). GraphPad Prism, version 9.5.0, was used for data analyses. Table S1 presents the row statistics (Mean, S.E.M, N) and statistical method used for the data present in the results section. We used the Shapiro-Wilk test to verify the data normality. PPI results were analyzed by repeated-measures two-way ANOVA, considering “PP intensity” (PP70, PP75, and PP80) as within-subjects variables and “experimental groups” (zero-hit + SE, zero-hit + EE, two-hit + SE, and two-hit + EE) as between-subjects variables. Mean % PPI, Y-maze, OFT, and SIT were analyzed by two-way ANOVA using “hit” (zero-hit or two-hit) and “environmental condition” (standard and enriched) as factors followed by Sidák’s multiple comparison test. Western blot was analyzed by one-way ANOVA followed by Dunn’s or Tukey's multiple comparisons tests. High-Resolution Melting curves were qualitatively analyzed. In all comparisons, P < 0.05 was considered statistically significant.

3.1 Environmental enrichment prevents schizophrenia-like behavioral alterations in male mice exposed to the neurodevelopmental “two-hit” model.

We evaluated the % PPI since reducing this phenomenon, which prepares the organism to deal with a potentially relevant stimulus, is a biomarker of schizophrenia ³³. We observed that animals exposed to the two-hit + SE presented significant PPI deficits in all PP intensities tested (Fig. 2A) compared with the zero-hit + SE group. The exposure to EE completely prevented PPI deficits in two-hit mice (zero-hit + SE vs. two-hit + SE: PP70: mean diff.: 59.73, 95.00% CI of diff.: 42.18 to 77.28, P = 0.0015; PP75: mean diff.: 57.17; 95.00% CI of diff.: 22.18 to 92.16, P = 0.0046; PP80: mean diff. 60.74, 95.00% CI of diff.: 40.98 to 80.50, P < 0.0001; two-hit + SE vs. two-hit + EE: PP70: mean diff.: -60.77, 95.00% CI of diff.: -92.39 to -29.14, P = 0.0017; PP75: mean diff.: -58.05; 95.00% CI of diff.: -93.01 to -23.10, P = 0.0040; PP80: mean diff. -60.72, 95.00% CI of diff.: -80.51 to -40.93, P < 0.0001).

The analysis of the mean value of %PPI by two-way ANOVA (Fig. 2B) considering the factors “hit” and “environmental condition” revealed a significant interaction [F (1, 34) = 70.70, P < 0.0001] with a significant main effect of “hit” [F (1, 34) = 40.48, P < 0.0001] and “environmental condition” [F (1, 34) = 42.52, P < 0.0001]. Indeed, the post hoc test revealed that the two-hit + SE group presented decreased mean %PPI compared with zero-hit + SE, which was completely prevented by EE condition (zero-hit + SE vs. two-hit + SE: mean diff. 59.21, 95% CI of diff. 42.80 to 75.63, P < 0.0001; two-hit + SE vs. two-hit + EE: mean diff. -59.85, 95% CI of diff. -75.12 to -44.58, P < 0.0001).

In the analysis of the traveled distance in the OFT (Fig. 3A and B), we observed no significant interaction between “hit” vs. “environmental condition”, but a significant main effect of “environmental condition” [F (1, 39) = 18.56, P = 0.0001]. This result revealed that mice exposed to EE condition, independent of hit exposure, tended to increase locomotor activity in the field, although not statistically significant.

Working memory evaluation by the Y-maze task (Fig. 3C) revealed that the exposure to two-hit + SE significantly decreased the % spontaneous alternation compared with zero-hit + SE. EE condition prevented the alterations in working memory function induced by two-hit (two-way ANOVA interaction: [F (1, 32) = 5.696, P = 0.0231]; Šídák's multiple comparisons test - zero-hit + SE vs. two-hit + SE, mean diff. 14.31, 95.00% CI of diff. 3.609 to 25.01, P = 0.0042; two-hit + SE vs. two-hit + EE, mean diff. -15.66, 95.00% CI of diff. -25.94 to -5.377, P = 0.0010).

In the SIT (Fig. 3D), we observed that the EE condition caused a slight, but not significant reduction of the social preference index of mice exposed to zero- and two-hit compared with SE animals (two-way ANOVA interaction: [F (1, 22) = 0.004177, P = 0.9491]; significant main effect of hit [F(1,22) = 5.9, P = 0.023]; and environmental condition [F(1,22) = 19, P = 0.0002]).

3.2 Environmental enrichment normalizes hippocampal expression of Iba-1 and increases the expression of DCX in mice submitted to the “two-hit” model of schizophrenia

Since inflammation regulates neurogenesis, it was recently reported that the brains of post-mortem patients with high inflammation schizophrenia presented greater gene expression of phagocytic microglia markers and reduced gene expression of immature neuron marker doublecortin (DCX) ³⁴. Our results showed that mice exposed to two-hit + SE presented higher hippocampal protein expression of the microglia inflammatory marker IBA-1 compared with the zero-hit + SE group. At the same time, the EE condition prevented this alteration (Fig. 4A). Regarding DCX (Fig. 4B), we observed higher hippocampal expression of this neurogenesis marker in animals exposed to EE, independent of hit exposure (IBA-1- Dunn's multiple comparisons test, zero-hit + SE vs. two-hit + SE mean diff. -14.20, P = 0.0265, two-hit + SE vs. two-hit + EE mean diff. 16.00, P = 0.0141; DCX - Tukey's multiple comparisons test, zero-hit + SE vs. zero-hit + EE mean diff. -1.836, 95.00% CI of diff., -3.522 to -0.1511, P = 0.0288, two-hit + SE vs. two-hit + EE mean diff. -1.834, 95.00% CI of diff. -3.629 to -0.03908, P = 0.0438).

3.3 Environmental enrichment promotes demethylation of the Slc6a4 gene in the hippocampus of mice submitted to the “two-hit” model of schizophrenia

Patients with schizophrenia present alterations in the 5-HTT gene (SLC6A4), being the functional polymorphisms of this transporter are linked to the susceptibility to this mental disorder ¹⁴.

As depicted in Fig. 5, the analysis of the normalized melting curves obtained for Slc6a4 revealed that this gene was partially methylated (50%) in all samples from the two-hit + SE group compared with the control group (zero-hit + SE). The EE condition effectively promoted the demethylation of the Slc6a4 gene in all samples from the two-hit + EE, with a return to the methylation pattern of the control group (0%).

The present study shows that the EE condition implemented from infancy to adulthood prevents behavioral and hippocampal molecular alteration induced by the two-hit model of schizophrenia. Besides this, the main novelty of our study is that the two-hit model simulates Slc6a4 gene methylation alterations observed in schizophrenia patients ³⁵. At the same time, EE condition mitigated hippocampal pro-inflammatory and alterations in Slc6a4 gene methylation.

Our study applied a 7-week EE protocol in mice submitted to a neurodevelopmental model induced by a first hit in neonatal life, whose molecular alterations are progressive along life ⁹. Notably, the duration of EE has an important impact on behavioral and molecular outcomes. In this regard, a 3-week EE implementation in adult mice improved aversive stimulus-based memory performances and reduced anxiety-like behavior, while a 5-week exposure caused no obvious alteration ³⁶. On the other hand, mice submitted to a maternal separation protocol from PNDs 1–21 and to EE condition from PNDs 23 to 65 (6 weeks duration) were protected against the effects of maternal separation, i.e., had lower anxiety levels and reversed spatial memory deficits compared with animals housed in standard conditions. Additionally, in this 6 weeks EE protocol, the protective effect of EE was associated with hippocampal increased expression of BDNF and synaptophysin ³⁷. Hence, EE protocols with longer durations seem more efficient in preventing or correcting long-term alterations induced by early-life adversities.

The effect of EE condition during puberty on the development of long-term behavioral alterations has also been investigated in other animal models of schizophrenia ^38,39. A recent study using an animal model based on repeated N-methyl-D-aspartate receptor (NMDAR) antagonist MK801 administration during adolescence observed structural excitatory/inhibitory imbalance significantly correlated with animal recognition memory deficits, which EE prevented. Additionally, these authors showed that the selective chemogenetic activation of axonal bouton of parvalbumin-expressing interneurons (PVIN) in the frontal association cortex mimicked the effects of EE on both synaptic plasticity and animal behavior. In contrast, selective inhibition of PVIN abolished EE's beneficial effects. The same behavioral and molecular alterations induced by EE and PVIN manipulation were observed in adolescent PV-Cre; ErbB4fl/fl mice ⁴⁰. It should be mentioned that PVINs are fast-spiking GABAergic interneurons that provide inhibitory control of cortical and subcortical circuits, thought to be a key alteration underlying schizophrenia ⁴¹. Furthermore, PVINs in the hippocampus are crucial in schizophrenia cognitive dysfunction ⁴². Importantly, PVINs are susceptible to oxidative and inflammatory CNS microenvironmental alterations, as observed in several animal models of schizophrenia ⁴³, including the ones based on perinatal immune activation.

In our results, we observed that EE could prevent hippocampal inflammatory changes, i.e., abolish the increase in IBA-1 protein expression induced by the two-hit model of schizophrenia while also increasing neurogenesis (observed by DCX protein expression). In the two-hit model, the first hit is exposure to PolyI:C, which induces innate immune cell activation via Toll-like receptors (TLRs). PolyI:C selectively stimulates TLR3 proinflammatory cytokine production ⁴⁴. PolyI:C exposure early in life induces microglial priming, a state in which the cells respond more to stimuli. In summary, early life exposure to PolyI:C may lead to changes in microglial state and function, priming these cells for subsequent immune challenges, not beneficial to brain function ⁴⁵, i.e., leading to behavioral alterations such as cognitive deficits.

Additionally, peripubertal stress may increase proinflammatory cytokines and contribute to a lasting pro-inflammatory state⁴⁶. These immune alterations influence critical neurotransmission systems relevant to schizophrenia, such as dopaminergic, serotonergic, and glutamatergic. Additionally, the enzyme indoleamine 2,3-dioxygenase (IDO) of the tryptophan/5-HT/kynurenine metabolism has its expression and activity increased by immune activation, influencing the serotonergic and glutamatergic neurotransmission via the metabolites quinolinic (an NMDAR agonist) and kynurenic acid (an NMDAR antagonist), a mechanism underlying schizophrenia ⁴⁷.

Many preclinical studies have evaluated EE's effects on neuroinflammation markers, but as far as we know, no one has used the two-hit model of schizophrenia. In the short term, EE was a stronger microglial and peripheral T cell modulator in C57BL/6 wild-type mice ⁴⁸. On the other hand, another study showed that long-term EE reduced the inflammatory cytokines and increased hypertrophy and ramification of IBA1 in the hippocampus, hypothalamus, and amygdala without altering microglial cell density ⁴⁹. Once more, considering these studies that relate time and the beneficial effect of EE, the literature report that EE's degree of preventive and therapeutic effects on schizophrenia abnormalities may vary depending on age and duration of exposure ^50,51.

Great attention has been directed to the role of 5-HT in short- and long-term memory, cognition, mood, impulse control, and motor performance in psychiatric disorders such as schizophrenia ⁵². Indeed, 5-HT modulates dopaminergic neurotransmission, the core neurotransmission system of schizophrenia pathophysiology and treatment ⁵³, being the combined blockage of D2 and 5HT2A receptors, the main mechanism of atypical antipsychotic drugs ⁵⁴. Deserves mentioning that the hippocampus has a great expression of the 5-HTT in the stratum oriens of area CA3 and, to a lesser extent, in the stratum oriens of CA1 and the stratum lacunosum moleculare of CA1-CA3 ⁵⁵.

Our results showed that the Slc6a4 gene was partially methylated (50%) in all hippocampal samples of the two-hit group. As far as we know, this is the first evidence of such alteration in a two-hit animal model of schizophrenia. Indeed, since the 5-HTT plays a critical role in regulating serotonergic function, it was observed that polymorphisms in this gene are related to psychosis, depressive-related symptoms, and suicidal behavior in patients with schizophrenia ^56,57. Furthermore, altered SLC6A4 gene methylation is observed in stress-related psychiatric disorders ⁵⁸. Regarding schizophrenia, lower SLC6A4 mRNA expression was observed in drug-naive first-episode patients, whereas antipsychotic treatment had no significant effect on the expressions of SLC6A4 mRNA over an 8-week follow-up ⁵⁹. We observed that EE could demethylate the Slc6a4 gene in our animal model. Hence, based on our results, we can infer that EE effects in Slc6a4 gene methylation may be of great relevance in preventing or as an adjunct to the mechanisms of antipsychotic drugs. Furthermore, our results are promising for developing epigenetic drugs (the so-called epidrugs) for being used as preventive strategies or pharmacological treatment of schizophrenia patients, alone or combined with environmental interventions.

This study has some limitations. Firstly, we used only male mice. Since schizophrenia presents a sex bias, using male and female mice would be important. Despite this, the disorder in males, as previously mentioned, has more severe symptoms and early onset. Additionally, males are more prone to developing neurodevelopmental disorders than females, probably due to sex differences in immune response ²⁴. Secondly, we did not evaluate 5-HTT protein expression.

In conclusion, the present study provides novel evidence on altered hippocampal Slc6a4 gene methylation in an animal model of schizophrenia induced by two-hit. Furthermore, a 7-week EE implementation could abrogate behavioral impairment, Slc6a4 gene methylation, and proinflammatory hippocampal alterations induced by the model, an important alternative for schizophrenia prevention. This study brings new insights into molecular-targeted schizophrenia therapies based on environmental manipulation and epigenetic-driven approaches to target schizophrenia prevention and treatment strategies.

Author contributions

Conceived and designed the study and analysis: D. S. M.; G. C. A. and C. L. M. F. Funding acquisition: D. S. M. Collected the data: G. C. A.;F. S. B.; G. S. V.; C. N. C. L.; F. E. R. S. and C. L. M. F. Contributed to data or analysis tools: D. S. M.; G. C. A.; F. S. B.; G. S. V.; C. N. C. L.; F. E. R. S. and C. L. M. F. Performed the analysis: D. S. M.; G. C. A.; F. S. B.; C. N. C. L.; F. E. R. S.; and C. L. M. F. Wrote the paper: D. S. M. and F. S. B. with inputs from other authors. Graphs and figures: F. S. B. All authors discussed the results, contributed, and accepted the final version of the manuscript.

Data availability statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Financial support

This study was financed by the following Brazilian agencies: Fundação Cearense de Desenvolvimento Científico e Tecnológico (FUNCAP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and, National Council for Scientific and Technological Development (CNPq).

Additional Information

The author(s) declare no potential conflicts of interest concerning this research article, authorship, and/or publication.

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No competing interests reported.

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Long-term environmental enrichment prevents schizophrenia-like abnormalities and promotes hippocampal Slc6a4 gene demethylation in mice submitted to a two-hit model

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1. Introduction

2. Material and Methods

2.1 Animals and ethical aspects

2.2 Study Design

2.3 Behavioral tests

2.4 Biochemical and methylation analysis

2.5 Statistical analysis

3. Results

3.1 Environmental enrichment prevents schizophrenia-like behavioral alterations in male mice exposed to the neurodevelopmental “two-hit” model.

4. Discussion

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